This application addresses broad challenge area "Stem cells" and specific area: "Understanding the Heterogeneity of Cancer and its Environment" (14-CA-102). Tumor stroma interactions are widely recognized to be crucial modifiers of the biology of cancer, but molecular components of this interface have remained largely undefined. In hematopoeisis, osteoblasts in the bone marrow provide a critical stromal "niche" that regulates the homeostasis of hematopoietic stem cells (HSCs). Leukemia "stem cells" (LSCs) also home and engraft in this osteoblastic niche. Unlike most tumor-stroma models, where the stromal component remains poorly defined, osteoblasts are a highly characterized population that can be used to define potentially novel regulatory components of the leukemia niche. Here, we propose to use an RNA-interference screen on the niche to define a compendium of molecules that act functionally in the LSC/osteoblast and the HSC/osteoblast interface. We have shown that murine LSCs (generated by the misexpression of the human MLL-AF9 translocation gene) do not survive in vitro in isolation, but can proliferate and form self-renewing "cobblestone" colonies when cocultured on monolayers of osteoblastic cells that partially recapitulate an ex vivo niche. We have generated co-cultures containing "red" LSCs and "green" osteoblasts (using DS-red and GFP expressing mice respectively). Using an arrayed shRNA library, we propose to knockdown individual genes in either the "red" LSCs or "green" osteoblasts and quantify cell number and cobblestoning function using imaging software previously developed in our labs. These hairpins will then be validated using functional assays and cross-annotated for HSC versus LSC effect. These experiments will provide a compendium of molecular pathways that regulate the interface between osteoblasts and LSCs/HSCs, thus creating a platform for testing these components in in-vivo studies. Such "niche-based" functional screening also represents a novel technological paradigm to explore tumor-stroma interactions in general. PUBLIC HEALTH RELEVANCE: Tumor-initiating stem cells do not live in isolation, but reside in highly regulated microenvironments termed "niches" created by stromal cells;identifying and eradicating these interactions represents an unexploited target for anti-cancer therapy, akin to destroying the "home" of a tumor cell. In the bone marrow, osteoblasts create one such niche for leukemia cells, although the molecular mechanisms that govern this interface are unknown. In this project, we will develop new technologies to evaluate thousands of genes to find those that are essential for the functional engagement of leukemia stem cells with osteoblasts in the hopes of targeting these interactions in the future.